This invention relates to a cell detachment method for detaching a cell during a culture (cultured cell) from a substrate.
In the case of culturing cells, two methods are mainly employed in general. Namely, the two methods are a method of culturing cells in a state where the cells are allowed to float in a culture solution and a method of culturing cells in a state where the cells are spread on a purpose-built substrate (cell). Of the two methods, the culture method using substrate is employed more frequently since it has advantages such as easy observation using a microscope and capability of culturing plural cells as one lot as compared to the culture in which the cells are allowed to float. More specifically, since the cells are spread on the substrate in the culture method using substrate, the cells are immobile and stable, thereby making it possible to easily observe a culture process with a microscope. Also, since it is possible to culture the plural cells in a state where they are attached to one another to perform intercellular signaling, it is possible to conduct culturing in a state which is similar to the natural state. Therefore, the culture method using substrate is widely used at present.
Referring to FIG. 10, in the case of conducting a culture by employing the culture method using substrate, each of cells S adheres to a substrate 30 and adjacent cells S. Such adhesion is caused because the cell S secretes an extracellular matrix S′, i.e. a protein substance which is an adhesive substance, on its surface upon contact with the substrate 30 or the adjacent cells S and because an adhesive force of the cell itself is increased inside the cell. As a result, the plural cells S adhere to the substrate 30 in a state where they are attached to one another as shown in FIG. 10. The cells S gradually proliferate while maintaining the above-described state to increase in number.
When the cells S have proliferated to achieve the required number of cells, the cells are no longer cultured on the substrate 30 and are generally detached at least once from the substrate 30 to be transferred onto a larger substrate to be cultured thereon. Also, in the case of taking out the cells S that have proliferated to achieve a certain number of cells, it is necessary to detach the cells from the substrate 30.
In order to perform the detachment of the cells, various methods have been employed. For example, there has been known a method of physically scraping off cells S that adhere to a substrate 30 by using a cell scraper 31 as shown in FIG. 11. Also, as shown in FIG. 12, there has been known a method of detaching cells S by separating the cells from one another by a chemical treatment using a reagent T and including a collagenase treatment and a trypsin treatment (see Patent Publication 1, for example).
Also, as shown in FIG. 13, there has been known a method of detaching cells S by using a cell culture sheet 32 provided on a substrate 30 (see Patent Publication 2, for example).
The cell culture sheet 32 is a stimulus-responsive substrate of which characteristics are switched between a state wherein the cells S adhere easily thereto and a state wherein the cells S hardly adhere thereto upon reception of stimulation such as a temperature, pH, and light. For example, during a culture of the cells S, the cell culture sheet 32 is brought into a state wherein the cells S adhere easily thereto. Then, the state is changed into the state wherein the cells S hardly adhere thereto by changing a temperature for detaching the cells S. Thus, it is possible to detach the entire cells from the cell culture sheet 32 due to the reduction in adhesiveness. Particularly, since this method enables to detach the entire culls, it is suitably used for a horny coat culture, a skin culture, and the like.
[Patent Publication 1] JP-A-2005-160370
[Patent Publication 2] International Publication No. 02/10349
However, the following problems are left unsolved in the conventional methods.
With the method of physically detaching cells using the scraper 31, a death rate of the cells S is high since the cells S are damaged by the scraper 31, and it is difficult to conduct a passage culture. The same problem has been found in the case of conducting the detachment by performing the chemical treatment using the reagent T.
Further, since the entire cells adhered to the substrate 30 are detached at once with the methods, the methods do not enable to detach only a desired cell S or desired cell group from the cells S and are difficult to operate.
The method using the cell culture sheet 32 enables the detachment with a reduced damage as compared to the physical detachment method and the detachment method by way of chemical treatment. That is, since the method enables the detachment with a temperature change which prevents the cells S from being damaged (the cells S are destroyed or a proliferation speed is remarkably reduced when the temperature change is out of lower limit critical solution temperatures of 0 to 80° C.), it is possible to reduce the damage of the cells S.
However, since the entire cells are detached by this method, the method does not enable to detach only a desired cell S or desired cell group from the cells S and is difficult to operate as is the case with the above-described methods.
Also, since the culture is conducted on a special culture substrate in which the cell culture sheet (ex. a temperature responsive polymer) 32 is provided on the substrate 30, such culture environment can be unsuitable for a certain type of cells S.
This invention has been accomplished in view of the above-described circumstances, and an object thereof is to provide a cell detachment method for detaching only a desired cell or cell group from plural cells cultured on a substrate without influencing on other cells.